Lactone compound of agricultural utility

ABSTRACT

Disclosed in the present invention are: a substance F-0368 or salts thereof having the following structural formula ##STR1## a method for production of the substance F-0368 or salts thereof, wherein an F-0368-substance-producing-microorganism belonging to a genus Kitasatosporia is cultured, and the substance F-0368 is isolated and collected from the cultured substance; a method for production of the substance F-0368 or salts thereof, wherein 3-methyl-4-lower alkoxy carbonyl-2(5H)-furanone represented by the following general formula is subjected to hydrolytic reaction ##STR2## (where: R represents a straight or branched lower alkyl group having 1 to 6 carbon atoms); a composition which consists essentially of the substance F-0368 or salt thereof, a diluent permissible in the agricultural chemicals, and a carrier or an excipient; a preventive agent for plant diseases such as rice blast disease and bacterial leaf blight disease, which consists essentially of an effective amount of the substance F-0368 or salts thereof as the active component, a diluent permissible in the agricultural chemicals, and a carrier or an excipient; a method for preventing plant diseases, wherein plants are treated with the preventive agent; a disinfectant for agricultural use to prevent plant disease such as rice blast disease and bacterial leaf blight disease, which consists essentially of an effective amount of the substance F-0368 or salts thereof, as the active component, a diluent permissible in the agricultural chemicals, and a carrier or an excipient; materials for agricultural use containing therein the substance F-0368 or salts thereof; a method for growing plants, wherein the plants are cultivated by use of the agricultural materials; and an F-0368-substance-producing microorganism (Kitasatosporia sp. F-0368; FERM BP-2259).

This invention relates to a novel substance identified as "F-0368". Moreparticularly, it is concerned with such "F 0368" substance useful asdisinfectant for agricultural purposes; process for its production;agricultural chemical composition containing such substance; preventiveagent for plant and vegetable diseases; disinfectant for agriculturaluse; materials for agricultural use; method for preventing diseases inagriculture using such disinfectant; method for growing plants usingsuch disinfectant; and F-0368 substance producing microorganism.

As the preventive agent against rice blast disease, there haveconventionally been known iso-protiolane, phthalide, IBP, probenazole,EDDP, and others; and, as the preventive agent against bacterial leafblight disease, there have been known phenazine oxide, probenazole,organic nickel compounds, and others.

Since the above-mentioned conventional chemicals are unable to displaytheir sufficient activity with a low dose, a relatively high dose ofsuch chemicals have been practiced so far, on account of which highpossibility of environmental pollutions in air, water, soil, and soforth have been apprehended, and also some of these chemicals wereharmful to human being and animals.

Therefore, the present invention aims at providing a novel substancewhich exhibits sufficient activity with a low dose, and possessexcellent preventive effect against various plant diseases such as, inparticular, rice blast disease and bacterial leaf blight disease.Further, the present invention aims at providing process for productionof such novel substance, agricultural chemical compositions containingtherein such novel substance, preventive agent for plant and vegetablediseases by use of such substance, disinfectant for agriculturalpurposes using such substance, materials for agricultural purposescontaining such substance, method for preventing diseases in agricultureusing such disinfectant, method for cultivating plants using suchdisinfectant, and microorganism producing such novel substance.

With a view to searching out useful agricultural chemicals, the presentinventors conducted research activities by separating a number ofmicroorganisms from various soil, isolating substances produced by themicroorganisms, and identifying the substances as obtained. As theresult, they discovered that a strain of ray fungus identified as"F-0368" belonging to a genus Kitasatosporia as separated from avegetable field in Nerima-ku, Tokyo, Japan had been able to accumulatein a culture medium a substance having excellent preventive effectagainst rice blast disease and bacterial leaf blight disease. Based onthis finding, the present inventors isolated the substance produced bythe microorganisms, and compared its physico-chemical properties withthose of known substances, as the result of which they confirmed thatthe substance was new, which had not been identified hitherto, and thuscompleted the present invention.

This novel substance of "F-0368" according to the present invention hasits physico-chemical properties as indicated below.

1) Structural Formula: ##STR3##

2) Melting point:

165° C. to 168° C. (measured by use of an automatic melting pointmeasuring device "METLER-FP1");

sublimating at a temperature ranging from 133° C. to 134° C. (measuredby use of a thermal analysis device "SHIMADZU TG-30").

3) Solubility to Solvent:

Readily soluble in methanol, ethanol and acetone; Soluble in ethylacetate and water; Non-soluble in chloroform, benzene and hexane.

4) Ultraviolet Ray Absorption Spectrum;

Indicating maximum absorption at 225 nm

    λ.sub.max.sup.MeOH (E.sub.1 cm.sup.1%)=225 nm (1070)

5) Distinction Among Basic, Acidic and Neutral:

Acidic substance

6) Color Rendering Reaction:

Positive: potassium permanganate

Negative: iodine, ninhydrin, ferric chloride, and2,4-dinitrophenylhydrazine

    ______________________________________                                        (7) Elemental Analyses:                                                                     C        H      O                                               ______________________________________                                        Measured Value (%)                                                                            50.63      4.25   45.10                                       [Theoretical Value (%)]                                                                       50.70      4.22   45.07                                       ______________________________________                                    

8) Infrared Ray Absorption Spectrum:

Infrared ray spectrum measured by the KBr method 3000, 2740, 2640, 2520,1730, 1670, 1450, 1430 1380, 1360, 1210, 1150, 1110, 1020, 890, 880,760, 710 (cm-⁻¹)

9) Nuclear Magnetic Resonance Spectrum (60 MHz): δ(ppm)

Measurement carried out in heavy methanol d₆ -DMSO 2.05(3H, t), 4.85(2H,q), 11.33(1H, s)

As is apparent from the above physico-chemical properties, the "F-0368"substance according to the present invention proves to be an acidicsubstance, hence it can be modified into a salt form. Examples of thesalt are as follows: alkali metal salts such as sodium salt, potassiumsalt, lithium salt, and so on; alkaline earth metal such as calciumsalt, magnesium salt, and so forth; ammonium salt; organic salts such astriethylammonium salt, and others; and complex salts of iron, copper,cobalt, and so forth.

The substance "F-0368" according to the present invention can beproduced from a strain of microorganism belonging to a genusKitasatosporia which is capable of producing the substance "F-0368".

As one example, the present inventors isolated from soil in a vegetablefield at Nerima-ku, Tokyo, Japan a actinomycetes belonging to a genusKitasatosporia having the following mycological properties, and theygave this strain or microorganism a nomenclature of Kitasatosporia sp.F-0368. This strain was deposited on Jan. 28, 1988 with FermentationResearch Institute, Japan, and assigned a deposit number of "FERMP-9845", which was transferred to an international deposition on Jan.26, 1989 under "FERM BP-2259".

The natural and artificial mutant strains of the above-mentioned strainof Kitasatosporia sp. F-0368 belongs, as a matter of course, to thegenus Kitasatosporia. Therefore, all the microorganisms capable ofproducing the substance "F-0368" according to the present invention canbe used, without exception, for the purpose of the present invention,hence they are included in the strains to be used in the presentinvention.

The mycological properties of the strain "F-0368" which produces thesubstance "F-0368" according to the present invention are as follows.

(I) Morpholoqical Properties

The strain of the actinomycete "F-0368" to be used in the presentinvention has a branched mycelium, of which the aerial myceliumextending in the air is fragmented and its terminal end forms a longchain of spores in number ranging from 5 to 20. The strain exhibitsparticularly favorable growth and formation of spores in a starch-agarmedium, yeast-malt-agar medium, and yeast-starch-agar medium. Itssubstrate mycelium assumes yellowish brown to brown in color, its aerialmycelium assumes white to gray in color. Observation through an electronmicroscope reveals that the terminal end of the spore which has beenbranched with respect to the principal axis indicate a rectus-flexibilisform, and, in a rare occasion, it shows a hook form. The spore has asmooth surface or an irregular rugose surface. It is cylindrical orelliptical in form with a dimension of 3 μm-4 μm×1.7 μm-2.0 μm. Nosporangium, sclerotium, and coremium were observed.

(II)Cultural Characteristics

Using various culture media, the properties of the strain "F-0368" wereexamined. The experiments were conducted in accordance with the methodas reported by E. B. Sharring et al. ("International Journal ofSystematic Bacteriology", Vol. 16, pp.313 to 340, 1966), with additionaluse of the well known culture media and experimental methods. The colortone was determined by use of "Color Harmony Manual", 4th Ed., byContainer Corp.)" as a standard color chart under a standard lightsource with a xenon lamp as the source of light, wherein, if acoincident color chart is found, a generic name is shown first and acolor chart code is subsequently shown together in a parenthesis. Unlessotherwise specified hereinafter, growth of the strain in the culturemedium was carried out in an agar plate culture for three weeks at atemperature of 28° C. The detailed results of the experiments arindicated in Table 1 below.

                                      TABLE 1                                     __________________________________________________________________________                            Color of                                                                            Color of                                                       Aerial   aerial                                                                              substrate                                                                           Soluble                                   Culture medium                                                                         Growth                                                                              mycelium mycelium                                                                            mycelium                                                                            pigment                                   __________________________________________________________________________    sucrose-nitrate-                                                                       poor  poor to  2db   none  none                                      agar medium    moderate (ivory)                                               (Difco, Czapek                                                                solution agar)                                                                glucose- moderate                                                                            good     7ba˜10dc                                                                      4ic   none                                      asparagine-    sparse   (pink (pastel                                         agar medium             tint˜                                                                         orange                                                                  orchid                                                                              suntan)                                                                 haze)                                                 glycerol-aspar-                                                                        poor  moderate 2db   13ba  none                                      tate-agar medium                                                                             sparse   (ivory)                                                                             (alabaster                                      (Difco, ISP-5)                tint)                                           starch-agar                                                                            good  abundant 5fe   3ni   none                                      medium (Difco,                                                                         sparse         (ashes)                                                                             (clove                                          ISP-4)                        brown)                                          nutrient agar                                                                          moderate                                                                            none     none  2fb   none                                      medium   slightly             (bamboo)                                                 good                                                                 tyrosine agar                                                                          moderate                                                                            moderate 10cb  3le   none                                      medium (Difco, (cottony) slightly                                                                     (orchid                                                                             (yellow                                         ISP-7          sparse   mist) maple)                                          yeast-malt-agar                                                                        abundant                                                                            abundant 5fe   3pl   none                                      medium                  (ashes)                                                                             (deep                                                                         brown                                                                         covert                                                                        brown)                                          oat meal-agar                                                                          poor˜                                                                         good     13cb  2ca   none                                      medium   slightly       (pearl                                                                              (light                                                   moderate       grey) ivory egg                                                                     shell)                                          yeast starch                                                                           moderate˜                                                                     good     10dc  2nl˜3gc                                                                       none                                      agar medium                                                                            good  sparse   (orchid                                                                             (covert                                                  sparse         haze) brown˜                                                                  light tan)                                      __________________________________________________________________________

III)Physiological Properties

1) Temperature range for growth (growth as at the end of the second weekin the maltose Bennet-agar medium, at a pH value of 7.3 beforesterilization and using a temperature gradient apparatus)

Appropriate temperature . . . 19.2° C. to 36.2° C.

Growable temperature . . . 14.0° C. to 42.3° C.

Liquefaction of gelatin (stab culture in glucose-peptone-gelatin medium)

20° C.: negative

27° C.: pseudo-positive

3) Coagulation and peptonization of milk (Difco, skim milk medium at 28°C. and 37° C. respectively)

28° C. peptonized: positive, coagulation: negative

37° C. peptonized: positive, coagulation: negative

4) Formation of melanine

tyrosine-agar medium: negative

melanine forming medium: negative

peptone-yeast-iron-agar medium: negative

tripton-yeast-liquid medium: negative

5) Dissolution of adenine, xanthine, hypoxanthine, and tyrosine adenine,xanthine, and tyrosine: negative hypoxanthine: positive

6) Salt resistance (in maltose-Bennet-agar medium+NaCl at 28° C.) Growthobserved upto 2% of the medium (no growth seen at 3%)

7) Capability of utilizing carbon source (in Difco, carbon utilizationagar medium at 28° C., at the end of second week)

positive: D-glucose, L-arabinose, D-xylose, rhamnose, salicin

negative: sucrose, D-fructose, raffinose, i-inositol, D-mannitol

(IV)Chemical properties

From hydrolyte of the whole cells, both LL-diaminopimelic acid andmeso-diaminopimelic acid were detected.

From the aerial mycelium on the agar plate, a large amount ofLL-diaminopimelic acid was detected, but mesodiaminopimelic acid wasdetected only slightly.

As the sugar component, there were detected galactose and mannose. Theprincipal menaquinone is MK-9 (H₆.H₈). The fatty acid composition is ofa branched type. The acyl type of the amino group in the muramic acid isthe acetyl type.

As described in the foregoing, the strain of microorganism F-0368according to the present invention has been identified as belonging tothe genus Kitasatosporia, since it possesses the genuine branchedmycelium; the aerial mycelium extending into the air is fragmented toform a long chain of spores; the amino acid composition in the wholecells contains both LL-diaminopimelic acid and meso-diaminopimelic acid;a large quantity of LL-diaminopimelic acid is detected from the analysisof diaminopimelic acid in the aerial mycelium; and the menanoquinecomponent is principally made up of MK-9 (H₆.H₈), hence the strain ofmicroorganism was identified as Kitasatosporia sp. F-0368.

The substance F-0368 according to the present invention can be producedby culturing an F-0368-producing-microorganism belonging to the genusKitasatosporia such as, for example, Kitasatosporia sp. F-0368 in aculture medium containing nutritive substances which ca be usuallyavailed by the microorganism, and by isolating and extracting thesubstance F-0368 from the culture broth. Examples of the nutritivesubstances which can be used for the purpose of the present inventionare: (i) as the carbon source, glucose, glycerol, sucrose, dextrin,starch, and so on; (ii) as the nitrogen source, soybean meal, wheatembryo, peptone, meat extract, yeast extract, corn steep liquor,ammonium salt, and so forth. Besides these substances, there may beused, depending on necessity, calcium carbonate, potassium chloride,magnesium sulfate, phosphates, and other inorganic salts.

As the method for culturing the microorganism, the liquid culture methodis suitable. The culturing conditions such as temperature, time, andothers are so selected that they may be adapted for the growth of themicroorganism used, and yet production of the substance F-0368 maybecome maximum. For instance, the culturing is carried out under anaerobic condition, and the cultivating temperature ranges from 25° C. to35° C., or it may perferably be 28° C. When the culturing is carried outunder aeration and agitation for a time period of from 92 to 144 hoursproduction of the substance F-0368 reaches its maximum.

For isolating and purifying the substance F-0368 produced andaccumulated in the culture broth, various methods which are usuallyemployed may be appropriately selected and used. For example, a methodof utilizing difference in solubility between the cultivated substanceand impurities, a method of utilizing difference in functional groups ofcompounds such as anion exchange resin, and a method of utilizingdifference in adsorptive affinity of silica gel, DIAION HP-20 (atradename for a product of Mitsubishi Kasei Corporation, Japan) and soforth can be used singly, or in combination, or in repetitive manner.

Verification of the active portion of the cultured substance can be doneby means of a silica gel thin layer chromatography (TLC).

One example of the method for isolating and purifying the substanceF-0368 is as follows: that is to say, while the substance F-0368 is alsopresent in the mycelia, it exists principally in the filtered liquid ofthe culture; accordingly, the mycelia is first removed by filtration orcentrifugal separation with addition of a filtration auxiliary to theculture broth, then the thus obtained culture liquid is rendered acidic(pH value of 2) with use of hydrochloric acid, and any coloring mattersare eliminated by means of a column chromatography of adsorbing resin(DIAION HP-20), to thereby elute the active substance.

Further, this active portion is collected and extracted by use of ethylacetate, in utilizing the property of organic solvents which is solubleunder acidity. Then, the extracted solution is cencentrated under areduced pressure, whereupon a crude extract of the substance F-0368 isobtained.

Subsequently, this crude extract is subjected to purification process bymeans of an adsorbing chromatography such as silica gel columnchromatography to collect the active portion, which is then concentratedand dried. As the result, there will be obtained crystals of thesubstance F-0368 in the form of a free acid.

Furthermore, the crystal in the free acid form as isolated can bemodified into the form of various salts by the conventional expedients.

As another method, the following synthetic method is also able toproduce the substance F-0368 according to the present invention.

3-methyl-4-lower alkoxy carbonyl-2(5H)-furanone [prepared by the methodof synthesis as described in "Tetrahedron, 31, (15) 1659" by S. W.Pelletier et al.] represented by the following general formula wasdissolved in, for example, water or a mixture of water and an alcoholsuch as methanol, ethanol, etc. in the presence of a solvent such ashydrochloric acid, sulfuric acid, tosylic acid, methanesulfonic acid,etc., and then the batch is subjected to reaction for a time period offrom 1 to 24 hours in general at a room temperature to 100° C.,whereupon the substance F-0368 as the end product can be obtained.##STR4## [where: R represents a straight or branched lower alkyl grouphaving 1 to 6 carbon atoms]

The substance F-0368 according to the present invention possessessufficient preventive activity with a low dose, and exhibits excellentpreventive effect against the rice blast disease and the bacterial leafblight disease.

That is to say, as the result of the experiments for preventing the riceblast disease, the substance F-0368 according to the present inventionexhibited not only such preventive effect by its spraying on the surfaceof leaves, but also indicated very excellent preventive effect, inparticular, in the experiments of soil treatment and spraying on thewater surface. Also, the substance exhibited excellent preventive effectagainst the bacterial leaf blight disease which is a bacterial diseaseof rice plant through its spraying on the water surface.

Such high activity of the substance F-0368 according to the presentinvention not only by its spraying o the surface of plant leaves, butalso by the soil treatment and its spraying on the water surface provesthat the substance possesses its remakable preventive effect.

Moreover, the excellent preventive effect exhibited by the substanceF-0368 according to the present invention against the rice blast diseasewhich is the disease of the rice plant due to the fungi, and against thebacterial leaf blight disease which is the disease of the rice plant dueto bacteria is a very peculiar phenomenon.

Incidentally, in its concentration of 500 μg/ml, the substance F-0368did not indicate its anti-microbial activity in vitro with respect tobacteria, yeast, and filamentous fungi.

Further, the substance F-0368 has low acute toxicity against mice, withthe consequence that the substance is less harmful to human being andanimals, hence it is highly excellent as the disinfectant foragricultural use.

As the disinfectants for agricultural use according to the presentinvention, which contain therein, as the active component, the substanceF-0368 and/or its salts, there may be enumerated the following examples:(1) disinfectants, in which the substance F-0368 is used as it is; (2)disinfectants obtained by diluting the substance F-0368 with use ofwater, solid powder, or other appropriate carries, and, depending onnecessity, adding an adjuvant such as spreading agent, etc.; or (3)various disinfectant formulations prepared by those methods which aregenerally adopted in the production of agricultural chemicals, whereinthe substance is mixed with various liquid or solid carries, and, ifnecessary, it is added with auxiliaries such as wetting agent, spreadingagent, dispersing agent, emulsifier, binder, and so forth, therebypreparing the substance in the form of water-dispersible powder, liquid,water-soluble powder, sol, emulsion, powder, granule, oil, and so on. Itis desirable that these various products be used properly depending onits purpose of use.

In preparing these formulations, the carrier to be used may be eithersolid or liquid. As those appropriate solid carriers, there may beenumerated: wheat bran; soybean meal, cellulosic powder, residue afterextraction of plants, bark and other vegetative carries; and clay, talc,kaoline, bentonite, China clay, calcium carbonate, diatomaceous earth,silicic anhydride, synthesized calcium silicate, and other inorganicsubstances. As those appropriate liquid carriers, there may beenumerated: water; aliphatic hydrocarbons such as ligroin, kerosene,mineral oil, and so on; aromatic hydrocarbons such as benzene, toluene,xylene, methylnaphthalene, and so forth; chloro-hydrocarbons such aschloroform, dichloroethane, and so on; alcohols such as methanol,ethanol, i propanol, n-butanol, and so on; ketones such as acetone,methylethyl ketone, cyclohexanone, and so on; nitrogen-containingorganic solvents such as ethanol amine, dimethyl formamide, and soforth; and various others.

With a view to improving the properties of the formulations orincreasing the biological effectiveness, it is also feasible to addvarious auxiliaries to the disinfectants for agricultural use accordingto the present invention. Examples are: various anionic or ionicsurfactans; high molecular weight compounds such as sodium alginate,carboxymethyl cellulose, methyl cellulose, polyvinyl alcohol, gumarabic, gelatin, casein, and so on; sulfite pulp waste liquor,"DORILESS" (a tradename for a product manufactured and sold by SankyoCo., Ltd., Japan), and so forth.

The disinfectant for agricultural use according to the present inventionmay contain therein various other active components which do not impairthe preventive effect of the substance F-0368. Such active componentsare, for example: fungicide, insecticide, herbicide, acaricide, plantgrowth regulator, nematicide, bacteriocide, repellent, attractant, andso forth.

According to the present invention, there is also provided materials foragricultural use, containing therein the substance F-0368 and/or itssalts. These agricultural materials can be obtained by mixing andblending the substance F-0368 and/or its salts with manure, culture soilfor rice plant seedling, culture soil for horticultural use, manure forhydroponic cultivation, and so forth.

In order to sterilize the disease fungi in agriculture with use of thesubstance F-0368 according to the present invention, the substanceF-0368 is incorporated in the above-mentioned disinfectants foragricultural use and materials for agricultural use, thereby killing thedisease fungi in agriculture.

For preventing the diseases in agriculture by use of the substanceF-0368, it is sufficient to carry out treatment by spraying and applyingthe substance F-0368 on the stems and leave of plants, or by absorptionof the substance from the roots of the plants through soil irrigation.

In case the substance F-0368 is to be used for sterilization andprevention of diseases in agriculture, the substance may be used in itsconcentration of from 10 ppm to 5,000 ppm in general, or preferably from100 ppm to 2,000 ppm, when the substance is to be sprayed on the surfaceof leaves.

In case the substance F-0368 according to the present invention is to beused for application to the nursery box and to the surface of water,concentration of the substance in the granule should be in a range offrom 0.1% to 50%, or preferably from 0.2% to 20%.

The quantity of application of the above-mentioned granules may begenerally in a range of from 30 g to 100 g per nursery box (having adimension of 30×60×3 cm containing therein about 5 l of soil), and from3 to 5 kg per 10 ares of paddy field.

As it is apparent from the foregoing explanations and will becomeevident from the preferred examples to follow hereinafter, the substanceF-0368 according to the present invention is excellent in its diseasepreventive effect with a low dose: in particular, it has remarkablepreventive effect against the blast disease and the bacterial leafblight disease of the rice plant, with low toxicity to human being andanimals, Accordingly, the disinfectant for agricultural use, thematerials for agricultural use, the method for disinfecting the diseasefungi and bacteria in agriculture and the method for preventing diseasesin agriculture according to the present invention ar of greatsignificance from the industrial standpoint. To add more, the substanceF-0368 can be efficiently produced with theF-0368-substance-producing-microorganism which belongs to the genusKitasatosporia.

In the following, it should be noted that the present inveniton is notlimited to these examples and experimental examples alone, but itincludes all possible embodiments which can be readily inferred from thefindings as already described in the foregoing.

In the accompanying drawing:

FIG. 1 shows an ultraviolet ray absorption spectrum of the substanceF-0368 according to the present invention;

FIG. 2 shows an infrared ray absorption spectrum of the substanceF-0368.

EXAMPLE 1 (Example of Producing Substance F-0368)

1 Culturing strain of microorganism Kitasatosporia sp. F-0368:

As the seed culture medium, use was made of the following composition:1.5% of dextrin, 3% of starch, 1% of soybean meal, 0.3% of meat extract,0.3% of polypeptone, 0.3% of yeast extract, 0.3% of calcium carbonate,and 0.1% of sodium chloride (the medium having a pH value of 7.0 beforesterilization).

70 ml each of the above-mentioned seed culture medium was separatelytaken into individual conical flasks, each having 500 ml capacity. Then,into each of these conical flasks, there was inoculated the strain ofmicroorganism of Kitasatosporia sp. F-0368, followed by shaken culturefor 72 hours a 28° C.

Subsequently, 1 l of this seed culture was transferred into a tankcontaining therein 100 l of production culture medium. This productionmedium was composed of: 1.5% of dextrin, 3% of starch, 1% of soybeanmeal, 0.3% of meat extract, 0.3% of polypeptone, 0.3% of yeast extract,0.3% of calcium carbonate, 0.1% of sodium chloride, and 0.2% ofantifoaming agent CA-123 (the medium having a pH value of 6.5 beforesterilization).

The culturing was conducted under aeration and agitation for 96 hours ata temperature of 28° C. The aerating quantity during the culturing was100 l/min. and the number of revolution for the agitation was 200 r.p.m.

2 Isolation and collection of the substance F-0368:

After completion of the culture, the culture broth (having pH value of8.4) was adjusted to have its pH value of 2.5 with hydrochloric acid,and then, as a filtration auxiliary, "Celite" (a tradename fordiatomeceous earth and related products of Johns-Manville ProductsCorp., U.S.A.) was added to the culture broth at a ratio of 4% forfiltration. Subsequently, approximately 50 l of filtered culture liquid(pH value of 2.5) was subjected to extraction for two times with use of50 l of ethyl acetate, after which the layer of ethyl acetate wasconcentrated under a reduced pressure, thereby obtaining approximately180 g of dry solid substance containing therein the substance F-0368.This dry solid substance was further dissolved into a samll amount ofethyl acetate, and subjected to in a silica gel column which had beenfilled in advance with ethyl acetate to develop this solid substancewith ethyl acetate, after which the active portion (about 1 l) wascollected and subjected to extraction using 1 l of distilled water,after the pH value thereof had been adjusted to 8.0 with sodiumhydroxide. In this way, the extracted active portion was transferredinto distilled water (pH value of 8.0) completely. Furthermore, thedistilled water was adjusted with hydrochloric acid to have its pH valueof 2.0, and the dry solid substance was subjected to a columnchromatograph (DIAION HP-20) having an adjusted pH value of 2.0 and thesubstance was developed with this acidic water (pH value of 2.0). Then,the active portion (approx. 1.5 l) was collected and subjected toextraction for two times with 1.5 l of ethyl acetate, after which thelayer of ethyl acetate was concentrated under a reduced pressure,thereby obtaining approximately 18 g of white planar crystals of thesubstance F-0368.

EXAMPLE 2

5 ml of 3N-HCl aqueous solution was added to 150 mg (0.96 mM) of3-methyl-4-methoxycarbonyl-2(5H)-furanone, and was stirred for threehours at a temperature of from 80° C. to 85° C. The reaction product wasthen extracted with ethyl acetate, and the solvent was fractionatedunder a reduced pressure. As the result, there was obtained an oilymatter. Ethyl acetate-n-hexane was then added to the thus obtained oilymatter to crystallize the same, whereby 110 mg of the substance F-0368was obtained with a rate of yield of 80.9%.

In the following, there will be shown examples of the formulations ofthe disinfectants for agricultural use according to the presentinvention. It should, however, be noted that kinds of additives to thesubstance F-0368 and their mixing ratio are in no way limited to theseexamples alone, but changes may be made in a wide range.

EXAMPLE 3 Dusting powder

3 parts by weight of the substance F-0368, 2 parts by weight of talc and55 parts by weight of clay were uniformly mixed in a mixer, and then themixed material was pulverized to obtain dusting powder.

EXAMPLE 4 Water-dispersible powder

20 parts by weight of the substance F-0368, 5 parts by weight of wettingagent (alkylbenzene sulfonic acid type), 2 parts by weight of whitecarbon, and 73 parts by weight of clay were uniformly mixed in a mixer,and then the mixed material was pulverized to obtain water-dispersiblepowder.

EXMPLE 5 Granule

20 parts by weight of the substance F-0368, 5 parts by weight of"TOXANON" (a tradename for a surfactant produced and marketed by SanyoKasei K.K., Japan), and 90 parts by weight of bentonite were granulatedby the wet-type extrusion method, and then the granules were dried andsieved to obtain the desired grain size.

In the following, experimental examples will be presented for the testsconducted on the effectiveness and acute toxicity of the disinfectantsfor agricultural use according to the present invention.

EXPERIMENT 1 Test for preventing rice blast disease (test by spraying onleaves)

Water-dispersible powder containing the substance F-0368 prepared inaccordance with Example 3 above was sprayed by use of a spray gun onrice plant (cv. "Nihonbare" at its 2 to 2.5 leaf stage), which wascultivated in a planting pot having a diameter of 9 cm, at a rate of 15ml per pot. After lapse of one day from the spraying, spore suspensionof the pathogenic fungus of this rice blast disease (i.e., Pyriculariaoryzae) was inoculated on this rice plant by spraying, which was thenplaced in an humidity chamber at a temperature ranging from 24° C. to26° C. and a relative humidity of 90% or above. After four days'passage, number of diseased leaf spots were counted to examine thepreventive effect of the disinfectant. The results are shown in Table 2below. It is to be noted that the value of preventive effect wascalculated from the following equation. ##EQU1##

                  TABLE 2                                                         ______________________________________                                                     Quantity of                                                                              Value of                                                           Effective  Preventive                                                         Component (ppm)                                                                          Effect (%)                                            ______________________________________                                        Water-dispersible                                                                            100          70                                                powder containing                                                                            500          75                                                the substance F-                                                              0368 of present                                                               invention                                                                     Non-treated    --            0                                                ______________________________________                                    

As is apparent from Table 2 above, the substance F-0368 exhibitsexcellent preventive effect against the rice blast disease by sprayingon the leaves.

EXPERIMENT 2 Test for preventing rice blast disease (test by applicationin nursery box)

50 g of the granule containing therein 1% of the substance F 0368, asprepared in accordance with Example 4 above, was uniformly scattered onrice plant (cv. "Nihonbare" at its 2.5 leaf stage) which was grown in anursery box with a dimension of 30 cm long, 60 cm wide, and 3 cm high.After lapse of 24 hours fron scattering of the granule, young seedlingwas cut out together with soil (1×1×3 cm), and transplanted to a Wagnerpot of 1/5000a with the soil having been attached to its root. Afterpassage of 40 days from the transplant, spore suspension of Pyriculariaoryzae, the pathogenic fungus of this rice blast disease, was inoculatedby spraying on this young seedling, and then it was placed in anhumidity chamber at a temperature of from 24° C. to 26° C. and arelative humidity of 90% or above, and kept therein. After lapse ofseven days, number of diseased spot was counted to examine the diseasepreventive effect of the granular disinfectant. The value of preventiveeffect was calculated in the same manner as Experiment 1 above. Theresults of the experiment are shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                                            Quantity of                                                                   of effective                                                                              Value                                                   Quantity  component   of pre-                                                 applied per                                                                             as applied per                                                                            ventive                                                 nursery box (g)                                                                         nursery box (g)                                                                           effect                                        ______________________________________                                        Granule containing                                                                        50          0.5         97                                        1% of F-0368 of                                                               this invention                                                                Probenazole 30          2.4         95                                        (granule containing                                                           8% of effective                                                               component)                                                                    Isoprothyolan                                                                             75          9.0         90                                        (granule containing                                                           12% of effective                                                              component)                                                                    Non-treated --          --           0                                        ______________________________________                                    

As is apparent from Table 3 above, the substance F-0368 exhibits moreexcellent preventive effect than the conventional disinfectants inrespect of the rice blast disease, by its application in the nurserybox.

EXPERIMENT 3 Test for preventive effect of rice blast disease (test byapplication onto water surface)

Each of three kinds of granule containing therein 1%, 2.5% and 5%,respectively, of the substance F-0368 prepared in accordance withExample 3 above was uniformly scattered onto the rice plant (cv."Nihonbare" at its fourth leaf stage) which has been grown in Wagner potof 1/10000a (depth of water filling of 3 cm) from above the surface ofwater.

After lapse of seven days from scattering of the granule, sporesuspension of Pyricularia oryzae, the pathogenic fungus of the riceblast disease was inoculated by spraying it onto this rice plant, whichwas then placed in an humidity chamber maintained at a temperature offrom 24° C. to 26° C. and a relative humidity of 90%. After passage ofseven days in the chamber, number of diseased leaf spots on upper twoleaves of the rice plant was counted to examine the preventive effect bythe granular disinfectant. By the way, the value of the preventiveeffect was calculated in the same manner as in Experiment 1 above. Theresults of the experiment are shown in Table 4 below.

                  TABLE 4                                                         ______________________________________                                                           Quantity of                                                          Quantity of                                                                            effective                                                            granule as                                                                             component  Value of                                                  applied per                                                                            as applied preventive                                                10a (kg) per 10a (g)                                                                              effect (%)                                      ______________________________________                                        Granule cont'ng                                                                           3          30         90                                          1% of F-0368 of                                                               this invention                                                                Granule cont'ng                                                                           3          75         95                                          2.5% of same                                                                  Granule cont'ng                                                                           3          150        98                                          5% of same                                                                    Probenazole 3          240        95                                          (granule contain-                                                             ing 8%)                                                                       Non-treated --          0          0                                          ______________________________________                                    

As is apparent from Table 4 above, the substance F-0368 according to thepresent invention possesses more excellent preventing effect than theconventional disinfectants in respect of the rice blast disease, by itsapplication onto the water surface.

EXPERIMENT 4 Test for preventive effect of rice bacterial leaf blightdisease (test by application onto water surface)

Each of two kinds of granule containing therein 2.5% and 5%,respectively, of the substance F-0368 preapred in, accordance withExample 3 above was uniformly scattered onto rice plant (cv. "Nihonbare"at its fifth leaf stage) which has been grown in the Wagner pot of1/10000a (with depeth of water filling of 3 cm) from above the watersurface.

After lapse of seven days from scattering of the granules, suspension ofXanthomonas oryzae, the pathogenic bacteria of this rice bacterial leafblight disease, was inoculated by needle into the leaves, and the thusinoculated rice plant was placed in a green house. After passage of 20days in the green house, length of the diseased lesion was measured toexamine the preventive effect of the granular disinfectant. The resultsare shown in Table 5 below. By the way, the value of preventive effectwas calculated by the following equation. ##EQU2##

                  TABLE 5                                                         ______________________________________                                                           Quantity of                                                                   effective                                                           Quantity as                                                                             component Value of                                                  applied per                                                                             as applied                                                                              preventive                                                10a (kg)  per 10a (g)                                                                             effect (%)                                       ______________________________________                                        Granule    4           100       85                                           cont'ng                                                                       2.5% of F-                                                                    0368 of this                                                                  invention                                                                     Granule    4           200       90                                           cont'ng 5%                                                                    of same                                                                       Probenazole                                                                              4           320       84                                           (granule                                                                      cont'ng 8%)                                                                   Non-treated                                                                              --          --         0                                           ______________________________________                                    

As is apparent from Table 5 above, the substance F-0368 according to thepresent invention also possesses excellent preventive effect againstbacterial leaf blight disease of rice plant by its application onto thesurface of water.

EXPERIMENT 5 Acute toxicity

Using JCL/ICR strain male mice (age 5 weeks; weight: 28±l g), a value ofLD₅₀ (determined by their mortality after two weeks from commencement ofadministration into the peritoneal cavity) revealed that the amount ofthe chemical was approx. 200 mg/kg.

We claim:
 1. A method of preventing plant diseases which consistsessentially of:applying an effective amount of the compound of theformula: ##STR5## to a plant.
 2. A method of disinfecting agriculturalareas to prevent plant diseases which consists essentially of:applyingan effective amount of the compound of the formula: ##STR6## to anagricultural area.
 3. A method of growing plants, consisting essentiallyof:cultivating plants in soil treated with the compound of the formula:##STR7##
 4. The method of claim 1, wherein said plant disease is riceblast disease of bacterial leaf blight disease.
 5. The method of claim2, wherein the agricultural area is disinfected against plant diseases.